The overall goals of the proposed research are to understand in molecular detail how human topoisomerase I functions to control the topological state of the DNA in the cell, and how tyrosyl-DNA phosphodiesterase (Tdp1) repairs the topoisomerase I-DNA covalent complexes that arise when the topoisomerase fails to religate the DNA after certain kinds of DNA damage and in the presence of the anticancer drug, camptothecin. A combination of biochemistry and X-ray crystallography will be used to study the conformational changes in the topoisomerase that accompany DNA binding and to elucidate the mechanism of the topoisomerase I religation reaction. A panel of GST fusions to various human topoisomerase I fragments will be employed to examine interactions of the topoisomerase with the HMG1 and Werner syndrome (WRN) proteins. A number of hypotheses will be tested concerning the functional significance of the interaction between human topoisomerase I and WRN. The identity of the nucleophile and the functions of other conserved active site residues in Tdp1 observed in the recently-solved crystal structure of the enzyme will be investigated by site-directed mutagenesis. Synthetic substrates that provide better leaving groups upon cleavage will be employed to identify the catalytic residue that acts as a general acid to protonate the leaving group when Tdp1 cleaves its DNA-protein substrate. A panel of substrates with different DNA structures and different lengths of topoisomerase I-derived peptides will be tested in kinetic experiments to determine the optimal substrate for Tdp1 cleavage. Finally, a system to study the in vivo fate of the topoisomerase I-DNA covalent complexes produced by the induction of a "toxic" topoisomerase I that cleaves, but fails to religate the DNA will be developed. The identification of intermediates along the pathway for the repair of these covalent complexes will provide important insights regarding how both the protein and the DNA moieties of the complexes are processed prior to cleavage by Tdp1. Information gained on the structure and function of Tdp1 may lead to the development of inhibitors of Tdp1 that could be used in combination anticancer drug therapy regimens with camptothecin.